Compositions and methods for regulating endogenous inhibitor of ATP synthase, including treatment for diabetes

Abstract

The present invention provides compositions and methods for altering mitochondrial ATP production, including fusion proteins comprising IF1 polypeptide-derived sequences and also including binding and functional assays exploiting IF1 interactions with ATP synthase. Also disclosed are methods for screening assays for a compound capable of reducing mitochondrial ATP hydrolysis and/or increasing mitochondrial ATP synthesis, including pharmaceutical compositions identified by such methods. The invention also provides methods for treating diabetes, and in particular, type 2 DM, using an agent identified according to the disclosed methods.

We claim:  
     
         1 . A method for identifying an agent that alters mitochondrial ATP production, comprising: 
 comparing (i) a level of binding of an endogenous inhibitor of ATP synthase to an ATP synthase subunit in the presence of a candidate agent to (ii) the level of binding of an endogenous inhibitor of ATP synthase to an ATP synthase subunit in the absence of the candidate agent, wherein an altered level of binding indicates that the agent alters mitochondrial ATP production.    
     
     
         2 . The method of  claim 1  wherein the endogenous inhibitor of ATP synthase is an IF1.  
     
     
         3 . The method of  claim 2  wherein the IF1 is a mammalian IF1.  
     
     
         4 . The method of  claim 3  wherein the mammalian IF1 is selected from the group consisting of a mouse IF1, a rat IF1, a rabbit IF1, a bovine IF1, a canine IF1, a non-human primate IF1 and a human IF1.  
     
     
         5 . The method of  claim 2  wherein the IF1 comprises a portion of an IF1 polypeptide, said portion comprising a polypeptide of less than 35 amino acids.  
     
     
         6 . The method of  claim 5  wherein the portion of an IF1 polypeptide comprises a polypeptide selected from the group consisting of the IF1 fragment 14-47 set forth in SEQ ID NO:29, the IF1 fragment 14-46 set forth in SEQ ID NO:67, the IF1 fragment 14-45 set forth in SEQ ID NO:66, the IF1 fragment 14-44 set forth in SEQ ID NO:65, the IF1 fragment 14-43 set forth in SEQ ID NO:64 and the IF1 fragment 14-42 set forth in SEQ ID NO:63.  
     
     
         7 . A method for identifying an agent that alters mitochondrial ATP production, comprising: 
 contacting, in the absence and presence of a candidate agent, an isolated IF1 polypeptide and an isolated mitochondrial ATP synthase, wherein the ATP synthase is capable of ATP synthesis, under conditions and for a time sufficient for ATP production to occur; and    comparing a level of ATP production by the ATP synthase in the presence of the candidate agent to a level of ATP production in the absence of the candidate agent, and therefrom identifying an agent that alters mitochondrial ATP production.    
     
     
         8 . The method of  claim 7  wherein the IF1 comprises a portion of an IF1 polypeptide, said portion comprising a polypeptide of less than 35 amino acids.  
     
     
         9 . The method of  claim 8  wherein the portion of an IF1 polypeptide comprises a polypeptide selected from the group consisting of the IF1 fragment 14-47 set forth in SEQ ID NO:29, the IF1 fragment 14-46 set forth in SEQ ID NO:67, the IF1 fragment 14-45 set forth in SEQ ID NO:66, the IF1 fragment 14-44 set forth in SEQ ID NO:65, the IF1 fragment 14-43 set forth in SEQ ID NO:64 and the IF1 fragment 14-42 set forth in SEQ ID NO:63.  
     
     
         10 . A method of treating diabetes comprising administering to a patient in need thereof an effective amount of a compound that (a) increases the synthesis of mitochondrial ATP in cells, (b) decreases the hydrolosis of mitochondrial ATP in cells, or (c) does both (a) and (b).  
     
     
         11 . The method of  claim 10 , wherein the compound is selected from the group consisting of a composition that inhibits one or more activities of IF1 and a composition that mimics IF1.  
     
     
         12 . The method of  claim 11  wherein the composition that mimics IF1 comprises a portion of an IF1 polypeptide, said portion comprising a polypeptide of less than 35 amino acids.  
     
     
         13 . The method of  claim 12  wherein the portion of an IF1 polypeptide comprises a polypeptide selected from the group consisting of the IF1 fragment 14-47 set forth in SEQ ID NO:29, the IF1 fragment 14-46 set forth in SEQ ID NO:67, the IF1 fragment 14-45 set forth in SEQ ID NO:66, the IF1 fragment 14-44 set forth in SEQ ID NO:65, the IF1 fragment 14-43 set forth in SEQ ID NO:64 and the IF1 fragment 14-42 set forth in SEQ ID NO:63.  
     
     
         14 . A method of identifying an agent useful for treating diabetes, comprising comparing (i) a level of ATP in a biological sample comprising at least one mitochondrion before contacting the sample with a candidate agent, to (ii) the level of ATP in the sample after contacting the sample with the candidate agent, wherein an increased level of ATP indicates the agent is useful for treating diabetes.  
     
     
         15 . The method of  claim 14  wherein the level of ATP in the sample is an intramitochondrial level of ATP.  
     
     
         16 . A fusion protein comprising a polyhistidine tag set forth in SEQ ID NO:1 fused to an IF1 comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS:12, 13, 29, 63, 64, 65, 66 and 67.  
     
     
         17 . A fusion protein comprising (i) a polyhistidine tag set forth in SEQ ID NO:1, which is fused to (ii) a cellular transport sequence comprising a tat sequence comprising any one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 10, 11 and 27, which is fused to (iii) a mitochondrial targeting sequence comprising an amino acid sequence set forth in SEQ ID NO:14, which is fused to (iv) an IF1 comprising any one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS:12, 13, 29, 63, 64, 65, 66 and 67.  
     
     
         18 . A fusion protein comprising (i) a polyhistidine tag set forth in SEQ ID NO:1, which is fused to (ii) a cellular transport sequence comprising a tat sequence comprising any one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 10, 11 and 27, which is fused to (iii) an IF1 comprising any one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS:12, 13, 29, 63, 64, 65, 66 and 67.  
     
     
         19 . A fusion protein comprising (i) a polyhistidine tag set forth in SEQ ID NO:1, fused to (ii) a cellular transport sequence comprising a tat sequence comprising any one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 10, 11, and 27.  
     
     
         20 . A fusion protein comprising (i) a polyhistidine tag set forth in SEQ ID NO: 1, fused to (ii) a cellular transport sequence comprising a tat sequence comprising an amino acid sequence set forth in SEQ ID NO: 10, which is fused to (iii) an IF1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO:29.  
     
     
         21 . An expression construct encoding a fusion protein of any one of claims  16 - 20 .  
     
     
         22 . A host cell comprising the expression construct of  claim 21 .  
     
     
         23 . A method of producing a fusion protein comprising culturing the host c ell of  claim 22  and recovering said fusion protein therefrom.